Free thyroxine is the generally accepted marker for the initial assessment of possible thyroid dysfunction: J. Clin. Invest., 45:133, 1966. Since approximately 0.05% of the total circulating thyroxine is physiologically active (i.e., free thyroxine), it is apparent that measurement of total thyroxine concentration only is not a completely satisfactory marker. Free thyroxine is however difficult to measure directly: Berger, S., and Quinn, J. L., Fundamentals of Clinical Chemistry, Tietz, N. W., Ed; W. B. Saunders, Co.: Philadelphia, 1976, Chapter 14 and Robbins, J., Thyroid Hormone Metabolism, Harlem and Orr, Ed., 1975, Chapter 1. The remaining circulating thyroxine is bound to protein, primarily thyroxine binding globulin (TBG): Robbins, J., and Rall, J. G., Proteins Associated with Thyroid Hormones, Physiol. Rev., 40, 415, 1960. Thus, the measurement of total circulating thyroxine concentration and of thyroxine binding globulin concentration provides a good assessment of the relative concentration of free thyroxine.
Various methods have been used to assess the level of thyroxine binding globulin concentrations. The oldest method is the T.sub.3 uptake study which measures unsaturated thyroxine binding globulin capacity: J. Clin. Endocrinol., 17:33, January, 1957 and J. Clin. Endocrinol., 25:39-54, 1965. T.sub.3 uptake assays use .sup.125 I labeled liothyronine (T.sub.3) to competitively bind to the free TBG sites and a secondary solid support (viz: resin, charcoal, etc.). After an incubation period and suitable separation technique (vis: washing, centrifugation, etc.) the remaining radioactivity on the solid support is counted. This radioactivity is inversely proportional to the unsaturated thyroxine binding globulin concentration.
Another methodology is a radioimmunoassay which utilizes antibody specific for thyroxine binding globulin. However methodology does not measure the binding ability of the thyroxine binding globulins: Clin. Chem. Acta., 87, (1978), 373-381.
A third methodology assesses the total thyroxine binding globulin capacity by equilibrating with a large excess of thyroxine to displace the endogenous thyroxine in the sample: U.S. Pat. No. 3,960,492 (1976).
Another methodology utilizes a thyroxine irreversible enzyme inhibitor conjugate, which binds by way of the thyroxine moiety of a site of unsaturation on the thyroxine binding globulin, thereby inactivating the irreversible enzyme inhibitor moiety. Thus, the greater the amount of unsaturated thyroxine binding globulin, the more conjugate that will be bound and the amount of irreversible enzyme inhibitor which will be available to inhibit the enzyme will be reduced. That is: increased amounts of thyroxine binding globulin will result in greater enzyme activity. Thus, intermixing enzyme and substrate to the enzyme and the above-described thyroxine irreversible enzyme inhibitor with serum permits determination of unsaturated thyroxine binding globulin in serum: U.S. Pat. No. 4,341,865.
Another methodology involves the use of fluorescent polarization immunoassay techniques to measure T.sub.3 uptake. However, this procedure involves separation steps as well as the use of antibody against T.sub.3 : U.S. Pat. No. 4,347,059.
It is known that thyroxine will bind to binding proteins other than thyroxine binding protein, in particular, albumin and pre-ablumin and therefore the presence of such proteins will influence free thyroxine levels. Fesco, et al, Clin. Chem., 28/6, p. 1325-1329 (1982).